Polyacrylamide gel electrophoresis has become one of the most frequently used techniques for the separation of biological macromolecules such as proteins, nucleic acids and polysaccharides. There is already a wide variety of equipment and methods for many types of high-resolution separation of these biological macromolecules for both analytical and preparative purposes. Two of the most widely used classes of separation methods involve: 1) separating protein molecules according to molecular weight using sodium dodecyl sulfate (SDS) denaturation, often referred to as “SDS-PAGE”, and 2) the separation of various types of amphoteric molecules (i.e. molecules with more than one ionizable chemical group) such as proteins using the principle of isoelectric focusing (IEF) on stabilized pH gradients where molecules migrate to a position in an electric field (the isoelectric point) where the pH environment provides a net zero charge on such an amphoteric molecule.
These two arts can be combined wherein a complex mixture of amphoteric molecules are initially focused in a one dimensional pH gradient means, such as a strip or tube of gel, to their component isoelectric points. The focused amphoteric molecules in the one dimensional pH gradient means are then subsequently separated by molecular weight using SDS denaturing electrophoresis in an orthogonal direction to the first dimension. This two-dimensional separation of complex mixtures using polyacrylamide electrophoresis is a powerful but difficult technique. It is often perceived as an art to be reserved for specialists in a biological research organizations who have cultivated the skills to create and implement first dimensional pH gradients in tubes or strips and to successfully equilibrate and transfer the resultant first dimensional gel onto a second dimensional gel using difficult manual transfer and alignment methods.
There is a great demand in everyday research, especially in protein based research, for the high resolution and parallel analysis that is afforded by this two-dimensional analytical approach. If this technique could be made convenient and easy to implement in a format that is familiar to the average laboratory researcher, its use would greatly increase. This is especially true for analysis of small regions of larger two-dimensional gels. The present invention discloses such a facile and convenient method of implementing two-dimensional gel electrophoresis in the popular research electrophoresis format known as a vertical “mini-gel”.